Anomalies in endocardial cushion formation lead to the most common congenital cardiac defects including septal defects (atria, ventricular and atrioventricular septal defects) and cardiac valve disease. We have previously shown that mice globally ablated for NM 2B developed cardiac structural defects such as double outlet of the right ventricle (DORV) and ventricular septal defects (VSD). Moreover, global introduction of the NM 2B motor-impairing mutation (R709C) into mice resulted in an additional cardiac defect, the abnormal enlargement of atrioventricular (AV) endocardial cushions. Endocardial cells play important roles in ventricular septal closure and cardiac cushion formation. Unlike the cardiac myocytes which show no detectable NM 2A, endocardial cells express both NM 2A and 2B. This study is designed to understand the role of NM 2 in regulating cardiac cushion development by conditional ablation of NM 2A and/or 2B from endocardial cells in mice using Nfatc1-cre. Ablation of either NM 2A (Anfatc1/Anfatc1) or 2B (Bnfatc1/Bnfatc1) alone in endocardial cells does not affect AV cushion remodeling. However compound ablation of NM 2B together with one allele of NM 2A (A+/Anfatc1;Bnfatc1/Bnfatc1) results in markedly increasing the thickness of the AV cushions at E14.5 and E17.5. A+/Anfatc1;Bnfatc1/Bnfatc1 AV cushions are less compact with a reduced cell density compared to control cushions. A+/Anfatc1;Bnfatc1/Bnfatc1 cushions also show reduced mesenchymal cell apoptotic activity, but no changes in their proliferative activity compared to controls. Compound Anfatc1/Anfatc1;B+/Bnfatc1 cushions show a moderate increase in cushion thickness, but not as severe as A+/Anfatc1;Bnfatc1/Bnfatc1 cushions, compared to control cushions. The data demonstrate that NM 2 (2A and 2B) is required for AV cushion remodeling to form thin and elongated normal cardiac AV valves. Since mice expressing motor-impaired R709C-NM 2B showed similar defects in cushion remodeling compared to compound endocardial NM 2A/2B ablated mice, these results provide further direct evidence that mutant NM 2B interferes with NM 2A in cardiac cushion remodeling.